Background/Objectives: Kidneys from marginal donors are more susceptible to ischemia–reperfusion injury (IRI). To diminish the deleterious effects of IRI, mitochondria should be preserved optimally between donation and transplantation. It is unknown which macronutrients are imperative to support mitochondrial function during normothermic preservation. The aim of this study is to investigate the effect of different macronutrient compositions on mitochondrial function during prolonged normothermic incubation of precision-cut kidney slices. Methods: Both porcine (n = 8) and human (n = 5) kidneys were studied. After warm ischemia and cold preservation, precision-cut kidney slices (PCKS) were made and incubated in different incubation media under normothermic conditions. PCKS were incubated with all different combinations of glucose, glutamine and/or fatty acids. At zero, 24 and 48 h, mitochondrial function and metabolite levels were assessed. Results: After 48 h of incubation, ATP levels were significantly higher with glucose and glutamine or fatty acids and glutamine, compared to incubation without nutrients (p = 0.001 and p = 0.003, respectively). Also, mitochondrial oxygen fluxes were higher with all nutrients compared to no nutrients (ADP stimulated basal respiration p = 0.040; proton leak p = 0.001 and maximal respiration p = 0.039). Conclusions: The addition of multiple macronutrients during prolonged normothermic incubation increases the mitochondrial function of PCKS. These data pave the way for optimising the nutritional support for ex vivo perfusion of marginal kidneys.

Background: Optimizing organ preservation techniques is imperative in the face of donor kidney shortage and high waiting list mortality. Hypothermic machine perfusion (HMP) has emerged as an effective method to improve graft function post-transplantation, particularly for deceased donor kidneys, prone to ischemia reperfusion injury (IRI). The perfusion solution includes glucose to support kidney metabolism; however, its effect on mitochondrial function remains unclear. The present study investigated the effect of glucose supplementation during 24 h of oxygenated HMP on mitochondrial function in porcine kidneys. Methods: After 30 min of warm ischemia, porcine slaughterhouse kidneys were preserved for 24 h using HMP with one of the following three solutions: the standard HMP preservation solution, University of Wisconsin machine perfusion (UW-MP) solution, which contains glucose; the solution used for static cold storage, University of Wisconsin cold storage (UW-CS) solution, which lacks glucose; or the UW-CS supplemented with 10 mmol/L glucose. Tissue and perfusate samples were collected before, during, and after perfusion for further analysis. Results: ATP production, mitochondrial respiration, and oxidative stress markers were not significantly different between groups. Glucose was released into the perfusion solution even from kidneys without exogenous glucose supplementation in the perfusate. Conclusions: These results suggest that kidney mitochondrial respiration does not depend on the presence of glucose in the HMP perfusion solution at the start of perfusion, underscoring the need for further exploration of nutrient supplementation and mitochondrial function in organ preservation strategies.